Polysaccharases for microbial exopolysaccharides

Microbial exopolysaccharides (EPS) are the substrates for a wide range of enzymes most of which are highly specific. The enzymes are either endoglycanases or polysaccharide lyases and their specificity is determined by carbohydrate structure with uronic acids often playing a major role. The presence of various acyl substituents frequently has little effect on the action of many of the polysaccharases but markedly inhibits some of the polysaccharide lyases including alginate and gellan lyases. The commonest sources of such enzymes can be either microorganisms or bacteriophages. These specific polysaccharide-degrading enzymes can yield oligosaccharide fragments, which are amenable to NMR and other analytical techniques. They have thus proved to be extremely useful in providing information about microbial polysaccharide structures and were routinely used in many such studies. Complex systems containing various mixtures of enzymes may also be effective in the absence of single enzymes but may be difficult to obtain with reproducible activities. Such preparations may also cause extensive degradation of the polysaccharide structure and thus prove less useful in providing information. Commercially available enzyme preparations have seldom proved capable of degrading microbial heteropolysaccharides, although some are active against bacterial alginates and homopolysaccharides including bacterial cellulose and curdlan.     

Microbial alginate production,modification and its applications

Alginate is an important polysaccharide used widely in the food, textile, printing and pharmaceutical industries for its viscosifying, and gelling properties. All commercially produced alginates are isolated from farmed brown seaweeds. These algal alginates suffer from heterogeneity in composition and material properties. Here, we will discuss alginates produced by bacteria; the molecular mechanisms involved in their biosynthesis; and the potential to utilize these bacterially produced or modified alginates for high-value applications where defined material properties are required.     

Role of substituents on the properties of some polysaccharides

This paper concerns the influence of the chemical structure on the physical properties of some polysaccharides. Especially, we proposed to discuss the role of the substituents on these properties. In some cases, non-carbohydrate substituents play a minor role on rheological properties in the presence of a salt excess as shown on xanthan and succinoglycan. The rheology of aqueous solution of these stereoregular polysaccharides is controlled by the conformation (helical conformation) whose stability is not largely influenced by these substituents. On the other hand, the interaction between galactomannan and xanthan depends on the presence of acetyl substituents on xanthan but also on the xanthan conformation. However, for polymers such as gellan, XM-6 or BEC 1615, complete deacetylation induces the ability to form physical gels in given thermodynamic conditions. The presence of carbohydrate substituents or short side chains was also examined. Especially in the gellan family, the role of position of substitution (position 3 on the glucose unit C or position 6 on the A glucose) was presented. It is concluded that the substituents giving the higher stability for the helical conformation (higher DeltaH and Tm values) also cause a lower salt sensitivity for the helical stability. The role of the substituents on the properties is also described for natural polymers and their chemically or enzymatically modified derivatives.     

Rheology and microstructure of solutions of the microbial polysaccharide from Pseudomonas elodea

Rheological studies of solutions and gels of the microbial polysaccharide from the organism Pseudomonas elodea have been combined with X-ray diffraction studies of fibres and pulsed electric-birefringence studies of dilute solutions, to investigate the conformation and interaction of the polymer molecules. Rheological data are suggestive of a locally rigid conformation for the biopolymer in solution. X-Ray diffraction studies suggest that the molecules adopt a three-fold helical structure. O-Acetyl substituents have been shown to inhibit the packing of these helices into crystalline domains. Studies of pulsed electric-birefringence suggest an extended, kinetically rigid structure in solution. Dissolving the polysaccharide in dimethyl sulphoxide inhibits the gelation and shear-thinning characteristics of aqueous solutions. Comparative studies of electric birefringence of solutions in water and dimethyl sulphoxide suggest that the differences in rheological properties may result from a change in molecular conformation.     

Bacterial alginate production: an overview of its biosynthesis and potential industrial production

Alginate is a linear polysaccharide that can be used for different applications in the food and pharmaceutical industries. These polysaccharides have a chemical structure composed of subunits of (1–4)-β-d-mannuronic acid (M) and its C-5 epimer α-l-guluronic acid (G). The monomer composition and molecular weight of alginates are known to have effects on their properties. Currently, these polysaccharides are commercially extracted from seaweed but can also be produced by Azotobacter vinelandii and Pseudomonas spp. as an extracellular polymer. One strategy to produce alginates with different molecular weights and with reproducible physicochemical characteristics is through the manipulation of the culture conditions during fermentation. This mini-review provides a comparative analysis of the metabolic pathways and molecular mechanisms involved in alginate polymerization from A. vinelandii and Pseudomonas spp. Different fermentation strategies used to produce alginates at a bioreactor laboratory scale are described.     

Occurrence, production, and applications of gellan: current state and perspectives

Bacterial exopolysaccharides (EPS) are products of biotechnology that are of high interest due to their rheological properties. This is the case of sphingans, a group of structurally related EPS secreted by members of the genus Sphingomonas. Among these, gellan is a multifunctional gelling agent produced in high yields by the non-pathogenic strain Sphingomonas elodea ATCC 31461. In its native form, gellan is a linear anionic EPS based on a tetrasaccharide repeat unit composed of two molecules of D: -glucose, one of L: -rhamnose and one of D: -glucuronic acid. The native gellan is partially esterified with acyl substituents (1 mol of glycerate and 0.5 mol of acetate) per repeat unit. Gellan has unique characteristics and has many applications, particularly in the food, pharmaceutical, and biomedical fields. This review summarizes current knowledge on the structure and properties of gellan and provides details about the biosynthesis of this exopolysaccharide. In addition, a highlight of the importance of gellan in industrial and medicinal applications is given.     

Comparative study of carrageenans from reproductive and sterile forms of <Emphasis Type="Italic">Tichocarpus crinitus</Emphasis> (Gmel.) Rupr (Rhodophyta,Tichocarpaceae)

A comparative study of the structure and properties of the sulfated polysaccharides (carrageenans) isolated from the vegetative and reproductive forms of the red alga Tichocarpus crinitus was performed. The polysaccharides were separated into the gelling (KCl-insoluble) and non-gelling (KCl-soluble) fractions by precipitation with 4% KCl. The total content of polysaccharides extracted from the reproductive form of the alga was 1.8-fold more than that extracted from the vegetative form, and in the first case, the gelling polysaccharides mostly accumulated. The gelling polysaccharides from the vegetative form have the highest molecular weight (354 kD). According to the results of FT-IR and 13C-NMR spectroscopy, the gelling polysaccharide fractions from both forms are kappa/beta carrageenans. The differences concern the content of the kappa- and beta-disaccharide units and the presence of a small content of the sulfated disaccharide segments (precursors of the kappa-carrageenans) in the polysaccharide from the reproductive form of the alga. The non-gelling polysaccharide fractions from both forms of the plant are mixtures of sulfated galactans with a low content of 3,6-anhydrogalactose.     

Microbial polysaccharides with actual potential industrial applications

The microbial polysaccharides reviewed include xanthan gum, scleroglucan, PS-10, PS-21 and PS-53 gums, polysaccharides from Alcaligenes sp., PS-7 gum, gellan gum, curdlan, bacterial alginate, dextran, pullulan, Baker's Yeast Glycan, 6-deoxy-hexose-containing polysaccharides and bacterial cellulose. Factors limiting the commercial potential of certain microbial polysaccharides such as availability, rheological properties, and polyvalency are outlined. The polysaccharides are classified according to their uses as viscosity-increasing agents and as gelling agents. A third category includes polysaccharides with specific applications such as tailor-made dextran and pullulan and polysaccharides used as substrates for the preparation of rare sugars. The difficulties encountered in development of a polysaccharide at the industrial level are pointed out.     

Gelatin vs polysaccharide in mixture with sugar

The structural behavior of a well-characterized gelatin sample has been revisited to investigate the morphology of its network in the presence of sugar. This was then contrasted with the corresponding properties of the gelling polysaccharides agarose, kappa-carrageenan, and deacylated gellan. Small deformation dynamic oscillation, differential scanning calorimetry in plain and modulated mode, visual observations, and transmission electron microscopy were used to identify the structural characteristics of the biopolymers from the rubbery plateau through the transition region to the glassy state. In contrast to the collapse of the polysaccharide gels at intermediate levels of co-solute, gelatin forms reinforced networks. The drop in polysaccharide network strength is accompanied by a decline in the enthalpy of the coil-to-helix transition, whereas the transition enthalpy is more pronounced in gelatin gels in accordance with their strengthening. Tangible evidence of the molecular transformations was obtained using microscopy, with polysaccharides disaggregating and dissolving in the saturated sugar environment. Gelatin, on the other hand, is visualized in an aggregated form thus producing a phase-separated topology with sugar.     

Genetics and engineering of microbial exopolysaccharides for food: approaches for the production of existing and novel polysaccharides.

Microbial exopolysaccharides (EPSs) are used in the food industry for their unique properties as viscosifiers, stabilisers, emulsifiers or gelling agents. In recent years, significant progress in the understanding of the genetics and biochemistry of microbial EPS synthesis by both Gram-negative and Gram-positive bacteria has been made. Biosynthesis pathways have been elucidated, and several of the genes involved have been characterised. This knowledge can now be applied to start EPS engineering or to improve EPS production.     

Fibre-diffraction studies of the extracellular polysaccharide from Pseudomonas elodea

X-ray fibre diffraction techniques have been used to probe the structure of the microbial polysaccharide from Pseudomonas clodca (gellan gum). The polymer adopts a threefold helical structure with an axial repeat of 0.94 nm. The polymer form weak elastic thermoreversible gels and on deacetylation forms rigid brittle gels. Deacetylation has been shown to enhance crystallinity. Equatorial reflections index onto a trigonal unit cell (a=b=1.64 nm.     

Polysaccharides and food processing

The rôle of polysaccharides during processing and for the quality of foods is discussed. Starch is the most important energy source for man. Most other polysaccharides are not metabolized for energy, but play an important rôle as dietary fibres. Pectins, alginates, carrageenans, and galactomannans are discussed as functional food additives in relation to their structure and their rheological behaviour, stability and interactions. Endogenous polysaccharides of fruits and vegetables and in products derived from them are responsible for such phenomena as texture (changes), press yields, ease of filtration and clarification, cloud stability, and mouth feel. To achieve desirable properties, the action of endogenous enzymes on polysaccharides must be inactivated and/or exogenous enzymes added as processing aids. This is also true for overcoming haze phenomena in clear juices or to break down undesirable microbial polysaccharides. Dough properties for bread baking can be improved by enzymic breakdown of a restrictive pentoglycan network. Network formation may come about by oxidative coupling of phenol rings of ferulic acid bound to hemicelluloses by ester links. Gels may be made by inducing oxidative coupling in natural or synthetic systems. Stagnation in development of new polysaccharide food additives is ascribed to difficulties in obtaining government approval for food use.     

Density distribution of calcium-induced alginate gels. A numerical study

Charged polysaccharides often form hydrogels in the presence of cations. In many applications the polymer network density distribution and associated physical properties are of major practical importance. Depending on the detailed conditions, the resulting gel density may vary from fully homogeneous to strongly inhomogeneous. We have established a simple set of coupled chemical reaction–diffusion equations to model the gelling process of calcium-induced alginate gels. The necessary algorithms for numerical solution of the resulting simultaneous parabolic differential equations have been developed both for one-dimensional models and three-ldimensional models with cylindrical or spherical symmetry. The algorithms make use of the Crank–Nicolson implicit finite difference method. The results of the numerical analyses of the gel formation can be divided into several different regimes depending on the physical and chemical parameters of the alginates and the cations. The numerical results are in good agreements with reported experimental results. © 1995 John Wiley & Sons, Inc.     

Microbial starch-binding domain

Glucosidic bonds from different non-soluble polysaccharides such as starch, cellulose and xylan are hydrolyzed by amylases, cellulases and xylanases, respectively. These enzymes are produced by microorganisms. They have a modular structure that is composed of a catalytic domain and at least one non-catalytic domain that is involved in polysaccharide binding. Starch-binding modules are present in microbial enzymes that are involved in starch metabolism; these are classified into several different families on the basis of their amino acid sequence similarities. Such binding domains promote attachment to the substrate and increase its concentration at the active site of the enzyme, which allows microorganisms to degrade non-soluble starch. Fold similarities are better conserved than sequences; nevertheless, it is possible to notice two evolutionary clusters of microbial starch-binding domains. These domains have enormous potential as tags for protein immobilization, as well as for the tailoring of enzymes that play a part in polysaccharide metabolism.     

Biofilm Formation by <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> Strains Differing in the Production and Rheological Properties of Their Exopolysaccharides

We evaluated the ability of several strains of the rhizobacterium Paenibacillus polymyxa, differing in the yield and rheological properties of their exopolysaccharides, to form biofilms on abiotic surfaces. Of these strains, P. polymyxa 1465, giving the highest yield of extracellular polysaccharides and the highest kinematic viscosity of the culture liquid and of aqueous polysaccharide solutions, proved to be the most active in forming biofilms on hydrophobic and hydrophilic surfaces. Enzyme-linked immunosorbent assay with rabbit polyclonal antibodies developed to isolated exopolysaccharides of P. polymyxa 1465 and 92 was used to detect P. polymyxa’s polysaccharidic determinants in the composition of the biofilm materials.     

Putrescine–polysaccharide conjugates as transglutaminase substrates and their possible use in producing crosslinked films

Putrescine (1,4-diaminobutane) was covalently linked to alginate and low-methoxyl pectin to synthesize new aminated polysaccharides. Both putrescine–pectin and –alginate conjugates, although the latter at higher concentrations, were found to be able to act as effective acyl acceptor transglutaminase substrates in vitro using both dimethylated casein and soy flour proteins as acyl donors. Monodansylcadaverine, a well known acyl acceptor transglutaminase substrate, dose-dependently counteracted the covalent binding of the aminated polysaccharides to the proteins. Putrescine–pectin conjugate was also tested to prepare, in combination with soy flour proteins, edible films in the presence of purified microbial transglutaminase. Characterization of the enzymatically crosslinked films showed a significant decreased water vapor permeability, with respect to the ones obtained with non-aminated pectin in the presence of transglutaminase, as well as improved mechanical properties, such as high extensibility. Possible biotechnological applications of hydrocolloid films containing putrescine–polysaccharide derivatives enzymatically crosslinked to proteins were suggested.     

Exopolysaccharides produced by a clinical strain of Burkholderia cepacia isolated from a cystic fibrosis patient

Burkholderia cepacia is an opportunistic pathogen involved in pulmonary infections related to cystic fibrosis. A clinical strain, BTS13, was isolated and the production of exopolysaccharides was tested growing the bacteria on two different media, one of which was rich in mannitol as carbon source. The primary structure of the polysaccharides was determined using mostly mass spectrometry and NMR spectroscopy. On both media an exopolysaccharide having the following repeating unit was produced: -->5)-beta-Kdop-(2-->3)-beta-D-Galp2Ac-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-Galp-(1--> This polysaccharide has already been described as the biosynthetic product of another Burkholderia species, B. pseudomallei, the microbial agent causing melioidosis. In addition to this, when grown on the mannitol-rich medium, B. cepacia strain BTS13 produced another polysaccharide that was established to be levan: -->6)-beta-D-Fruf-(2-->. The content of levan was about 20% (w/w) of the total amount of polymers. The ability of B. cepacia to produce these two exopolysaccharides opens new perspectives in the investigation of the role of polysaccharides in lung infections.     

Location and identity of the acyl substituents on the extracellular polysaccharides of Rhizobium trifolii and Rhizobium leguminosarum

The basic structures of the extracellular polysaccharides of Rhizobium leguminosarum and Rhizobium trifolii were found to be identical, but their acylation patterns differ. Liquid hydrogen fluoride at −40° degrades the two polysaccharides to a series of oligosaccharides representing the repeating units of the polysaccharides and their higher homologs. At −23°, it degrades the polymers to a mixture of oligosaccharides from which a tetrasaccharide constituting a unit of the backbone of the polysaccharide, and a trisaccharide representing all but the non-reducing terminus of the side chain, could be readily purified. The location and identity of the acyl substituents were determined by ¹H-n.m.r. spectroscopy, methylation analysis, and f.a.b. mass spectrometry. The unusual substituent d-3-hydroxybutanoate was found esterified to O-3 of a terminal 4,6-O-pyruvic acetalated d-galactose in both strains of R. leguminosarum, and in one of the three strains of R. trifolii tested. All of the strains tested contained a 3-O-acetyl substituent on the (1→4)-β-d-glucopyranosyl residues in the backbone of the polysaccharide. Only the R. leguminosarum polysaccharides contained a combination of 2- and 3-O-acetyl groups on the branching sugar of the backbone of the polymer.